Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/04—Preparations for care of the skin for chemically tanning the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/443—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin

Definitions

• This invention relates to means for the treatment of skin disorders and more particularly to those disorders requiring stimulation of melanocyte proliferation.
• the invention is of especial application to the treatment of vitiligo.
• Vitiligo is a common skin pigment disorder characterised by the development of patchy depigmented lesions.
• Current treatments which include the use of photosensitisers (eg psoralens) with UVA radiation (PUVA), corticosteroids or skin grafting have low success rates and are generally accompanied by unpleasant side effects.
• Vitiligo has a highly detrimental impact on the emotional well-being of the sufferer, the disfiguring effects of the disease being compounded by the absence of a suitable treatment.
• vitiligo patches are not believed to contain melanocytes (pigment producing cells), a reservoir exists in hair follicles in vitiliginous skin. Thus activation of hair follicular melanocytes is a crucial process in the repigmentation of vitiliginous skin.
• Certain plant remedies have been employed for the treatment of vitiligo for a long time and in many cases have given positive results in small scale studies.
• Psoralens which are employed in the modem PUVA and khellin in KUVA therapy were originally derived from plant sources ( Psoralea corylifolia L and Ammi visnaga respectively) used in traditional remedies for vitiligo.
• these therapies rely on the use of UV irradiation for their efficacy, which is associated with the aetiology of skin cancer.
• the fruit of black pepper ( Piper nigrum L. ) and long pepper ( Piper longum L. ) are both important medicinal herbs in Ayurvedic and Unani (traditional Indian) medicine systems, in which remedies generally consist of mixtures of herbs.
• a wide range of the medicinal uses of black pepper have been documented by Kirtikar and Basu (Indiam Medicinal Plants, 2 nd Edition, Vol. 3, (1935) pages 2128 - 2135), including its use in the treatment of leucoderma. Black pepper has also been implicated as a possible adjunct to Vernonia anthelmintica in the treatment of leucoderma (Indian Medicinal Journal, Vol. 1, 3 rd Edition, (1982) 1267 - 1270).
• compositions comprising black pepper, ginger and pipali have been used in the treatment of vitiligo (Ancient Science of Life, Vol. IX, No. 4 (1990) 202 - 206); however, the specific therapeutic action of black pepper in this orally administered composition has not been established.
• the compounds and compositions of the invention are able to stimulate the proliferation of melanocytes without significant adverse side effects.
• the present invention addresses that need.
• the present inventors have screened 30 herbal extracts, which have been used traditionally in the treatment of vitiligo using a cell culture system to evaluate whether they have the ability to directly stimulate the proliferation of melanocytes. It has been surprisingly found that components of Piper nigrum L. fruit possess melanocyte proliferant activity. In particular, it has been found that the component piperine is able to significantly stimulate the production of melanocytes.
• Piperine has also been reported to occur in other Piper species ie. P. acutisleginum, album, argyrophylum, attenuatum, aurantiacum, betle, callosum, chaba, cubeba, guineense, hancei, khasiana, longum, macropodum, nepalense, novae hollandiae, peepuloides, retrofractum, sylvaticum .
• Pharmaceutical compositions containing piperine have been used in the treatment of tuberculosis and leprosy (EP 0 650 728). It has also been suggested that piperine is able to enhance the bioavailability of the other constituents of a pharmaceutical composition (WO 96/25939).
• a first aspect of the invention provides the use in the preparation of a medicament for stimulating the proliferation of melanocytes of a compound of formula (I) in which
• De-pigmentation of the skin may arise, for example, from scar tissue formed as a result of a skin trauma such as burn or other skin lesion or may be due to skin conditions such as vitiligo.
• Compositions comprising piperine or an active analogue or derivative thereof may therefore be used to re-pigment post traumatised de-pigmented skin as well as conditions such as vitiligo.
• the medicament may be in the form of a solid powder; a paste, ointment or cream; a tablet or capsule; or a solution.
• the piperine or active derivative or analogue thereof may be administered by oral, topical, intravenous or subcutaneous (intramuscular) routes. It is preferred that the medicament is applied topically to the depigmented area of the skin.
• Pharmaceutical compositions used as medicaments are also included in the scope of the invention. Such pharmaceutical compositions comprise a compound of formula (Ia) and a pharmaceutically acceptable carrier.
• Compounds of formula (Ia) are the same as those of formula (I) but exclude piperine and its Z,Z, Z,E and E,Z steroisomers.
• a compound of formula (I) in the preparation of a medicament for re-pigmententing post traumatised depigmented skin.
• post traumatised de-pigmented skin it is to be understood to mean the skin formed during the healing process that occurs after a skin trauma such as a burn or a lesion.
• compositions may be administered orally, topical administration is preferred.
• Stimulation of melanocyte proliferation may also be used to enhance or promote the natural colouring of the skin and compositions comprising a compound of formula (I) can be used as tanning agents.
• the enhancement of natural skin colour may be used for prophylactic or cosmetic purposes.
• a further aspect of the invention provides a method for cosmetically enhancing or promoting the colouring of the skin comprising the administration to a person desiring such an effect of a cosmetically effective amount of a compound of formula (I).
• Administration may be by oral or topical routes.
• the compounds of formula (Ia) have not hereto before been used for therapeutic purposes and a further aspect of the invention provides a compound of formula (Ia) for use in the stimulation of melanocyte proliferation.
• the invention also provides a compound of formula (Ia) for use in therapy.
• Therapeutic uses include the re-pigmentation of de-pigmented skin as well as the promotion or enhancement of skin colouration.
• Piperine has also been found to inhibit the proliferation of melanoma cells.
• the compound of formula (I) may also be used in the treatment of skin cancer.
• a still further aspect of the invention provides the use of a compound of formula (I) in the preparation of a medicament for treating or preventing skin cancer in a human or non-human patient. Treatment may be effected through the administration to said patient of a therapeutically effective amount of a compound of formula (I).
• a further aspect of the invention provides the use of a compound of formula (I) in the preparation of a medicament for the treatment of skin cancer.
• the compound of formula (I) may be administered by oral or topical routes.
• Suitable dosage forms may be any of those discussed herein above.
• the naturally occurring compounds may be extracted from suitable plant sources or synthesised using methods known to a skilled person (see, for example, Chapman and Hall, Combined Chemical Dictionary on CD-Rom, Release 1:1 (1997) and The Merck Index (1983), 10th edition. Publ. Merck and Co, Rahway, USA. PP. 1477-1078 (except compounds 2 and 3)). All compounds except 6, 11, 13, 15 and 16 occur in P. nigrum or other Piper species (10 and 12).
• Compound 6 is prepared by hydrogenation of piperine over Adams catalyst using known methods (see for example Banerji A, Bandyopadhyay D, Sarkar M et al (1985). Structural and synthetic studies on the retrofractamides - amide constituents of Piper retrofractum , Phytochemistry, 24, 279-284).
• Compound 11 is prepared by methanolysis of piperine using sodium methoxide.
• Compound 16 is prepared from the reaction between piperidine and E,E-hexadienoyl chloride.
• the active compounds may be formulated for topical use in the form of creams, soft paraffin or lotions.
• Aqueous cream BP or Yellow Soft Paraffin BP may suitably contain the active at 0.03-3.0 mg % w/w or an equivalent amount of plant extract.
• a suitable lotion is typically prepared from 20% glycerol and 80% ethanol in purified water and contains 0.03-3.0 mg % w/w of the active material.
• These topical formulations may also contain penetration enhancers such as oleic acid, propylene glycol, ethanol, urea, lauric diethanolamide or azone, dimethyl sulphoxide, decylmethyl sulphoxide, or pyrrolidone derivatives.
• Liposomal delivery systems may also be used.
• compositions for oral formulation include tablets or capsules containing 1.5-150 mg active or equivalent amount of plant extract.
• Piper nigrum L. fruit black pepper, Piperaceae
• Piperaceae originally from India
• the rest of the herbs were either supplied by East-West Herbs, Kingham, Oxon, UK or by Cipla Ltd, Mumbai, India.
• the powdered dry herb (10 g) was heated to boiling in distilled water (100 ml) and allowed to boil for 10 min, using a hot plate as heat source.
• the plant material was filtered off under vacuum through filter paper (Whatman), and the filtrate freeze-dried.
• mice melan-a cell line (passage number 18-24), a first known line of non-tumorigenic pigmented mouse melanocytes were maintained in a flask (Costar, Cambridge, MA, USA) using RPMI 1640 (ICN, Costa, Mesa, CA, USA) as a basic medium.
• RPMI 1640 ICN, Costa, Mesa, CA, USA
• subconfluent melan-a cultures were trypsinized (0.25% trypsin at 37°C for 5-10 min) and inoculated with a repeater-pipettor (Finn pipette, Labsystems, Finland) into 96-well microtiter plates (Costar, Cambridge, MA, USA).
• the plates were incubated at 37°C in a 10% CO 2 , 90% air humidified atmosphere for the stated length of time.
• an SRB assay was performed. Briefly, cells attached to the bottom of the plate were fixed by addition of cold trichloroacetic acid (TCA, 4°C, Aldrich, Dorset, UK) on the top of the growth medium (final TCA 20% w/v).
• TCA cold trichloroacetic acid
• the plate was placed at 4°C for 1 hour before being gently washed five times with tap water. It was allowed to dry in air, or aided with a hair dryer to speed up the drying process, then 50 ⁇ l of 4% w/v SRB dissolved in 1% acetic acid in water was added to each well for 30 min.
• incubation conditions Prior to testing the herbal extracts, optimal culture conditions were established.
• the variable factors regarding incubation conditions include foetal bovine serum (FBS) concentration, initial cell seeding density and incubation period.
• FBS foetal bovine serum
• initial cell seeding density 1, 2, and 5% FBS were used to culture the melan-a cell line, the growth pattern with each concentration of FBS was monitored by SRB assay.
• SRB assay For the determination of optimum cell seeding density, a series of initial seeding density of 0.15 to 1.2 x 10 4 cell per well of melan-a cells were plated into 96-well plates with 5% FBS and 20 nM tetradecanoyl phorbol acetate (TPA) supplemented growth medium.
• TPA tetradecanoyl phorbol acetate
• FIG. 1 shows the growth curves of melan-a cell line at three different concentrations of FBS. Neither 1% nor 2% FBS supplemented medium was able to maintain cell survival; cell numbers declined significantly in 4 days of culture. However, 5% FBS was capable of keeping melan-a cell line alive with only a small increase in cell numbers observed over 4 days. TPA (20 nM) was able to cause further proliferation in the presence of 5% FBS indicating that cells were capable of responding to mitogenic stimuli at 5% FBS.
• the initial plating density of 6 x 10 3 cells/well exhibited exponential growth, and after 4 days of culture, the OD reading increased to a value of about 0.4. Since the higher OD values are associated with greater precision and accuracy, it was determined that the initial inoculation of 6 x 10 3 cells/well was the optimum density for the herbal test experiment. For the simplicity of the experiment, harvesting time was day 4 since the cells at this stage was not confluent and after 4 days, growth medium tended to become depleted and replacement was necessary for the further growth.
• Table 1 shows the results of the preliminary screening of 30 aqueous herbal extracts on the proliferation of melan-a cell line.
• Crude extracts of Astragalus membranaceous (Fisch.) Bunge, unripe Citrus reticulata Blanco, Dictamnus dasycarpus Turcz., Ophiopogon japonicus (Thunb.) Kergawe, Piper nigrum L., Poria cocos (Schw.) Wolf and Tribulus terestris L. were observed to stimulate melanocyte proliferation, sometimes even at the lowest dose level of 10 ⁇ g/ml. Other extracts either had no significant effect or were cytotoxic. Among these positive responses, that of Piper nigrum L.
• Piper nigrum extract at 0.01 and 0.1 mg/ml was the most pronounced. Piper nigrum extract at these two concentrations not only strikingly enhanced cell growth, but this extract also altered the cell morphology. In the presence of Piper nigrum extract, the cellular bodies were smaller, with more and longer bipolar or polydendritic processes, an effect similar to that observed with TPA.
• Piper nigrum fruit extract was tested on a new batch of melan-a cell line with the culture in microplates extended to 8 days.
• the effects of Piper nigrum extract on the growth of melan-a cell line were evaluated by SRB assay.
• Figure 3 shows that the result of the significant proliferant effect brought about by the Piper nigrum extract was even more marked on the extension of the incubation period to 8 days of culture, the growth was 272% of the control (cells only). Microscopically, the morphology of the cells was altered as those seen in the preliminary experiments.
• Melan-a cells were plated in petri dishes ( ⁇ 35 mm, Nunclon, Denmark) with a plating density of 2 x 10 4 /ml and Piper nigrum extract at concentrations of 0.01 and 0.1 mg/ml. A negative control (cells in medium only) and positive TPA (20 nM) control were also set up. After 4 days the cells in each dish were harvested and counted with haemocytometer.
• SRB assay indirectly estimates cell number through protein staining and spectrophotometric measurement.
• a direct cell counting with haemocytometer method was employed. Table 2 shows the cell numbers in the presence of Piper nigrum extract and 20 nM TPA. Cell number under the influence of Piper nigrum extract at 0.01 and 0.1 mg/ml were increased significantly compared to control, but less than that with 20 nM TPA. This result is consistent with the finding in 96-well microplate SRB assay.
• piperine has a highly selective effect on the growth of a panel of cell types, since it only stimulates the mouse melanocytes (melan-a, melan-c), human melanoblasts (FM21E), human foetal melanocytes (FM 21E) and the mouse fibroblast SC1 cell lines at the concentration tested.
• the SC1 cell line may have a particular sensitivity to TPA due to the way in which it has been derived, i.e. it has been cultured in the presence of TPA.
• piperine has either no effect or a cytotoxic effect on other cells. This result implies that piperine may have desirable specificity index for the proliferation of melanocytes in culture and is not a general mitogen.
• TPA a well known PKC activator and a tumour promoting agent
• Figure 5 shows the effect of RO-31-8220 on the survival and growth of melan-a cell line in the presence or absence of piperine and TPA.
• RO-31-8220 alone did not have significant cytotoxic effect to the cells at the concentrations up to 100 nM.
• the proliferant effects of piperine, and TPA (as indicated by the Y axis values) on melan-a cells were effectively inhibited by the presence of RO-31-8220 at the concentrations of 0.1 - 100 nM. It thus appears that piperine and TPA exert their proliferant effects through the activation of PKC cell signalling pathway.
• piperine The selectivity of piperine on the growth of a panel of cell types has also been tested. It was found that piperine possessed a fairly high specificity and selectivity towards melanocytes, since it significantly stimulated the growth of melan-a, melan-c and FM21E melanoblasts and FM21E melanocytes in culture, whereas it did not stimulate all other cells apart from a TPA-sensitive fibroblast cell line. Piperine was observed to have inhibitory effects on B16 mouse melanoma cell line which is syngeneic with melan-a cells. Thus piperine may be a specific stimulant for the proliferation of melanocytes in vitiliginous skin without the risk of stimulating melanoma cells.
• FIG. 6 shows the effect of piperine on the growth of human melanoblasts in vitro.
• Piperine at the concentrations of 1, 10, 100 ⁇ M was found to cause significant stimulation to human melanoblasts in a dose response manner, with 34% more cell yield compared to vehicle control when the culture was exposed to 100 ⁇ M piperine in culture for 5 days.
• TPA a well-known melanocytic growth-stimulating agent, was also able to cause significant cell growth at tested concentrations, with over 50% of more cell yield observed when the culture was exposed to 20 nM for 5 days.
• piperine was consistently observed to induce significant cell growth at the concentrations ranging from 5 - 100 ⁇ M; these stimulatory effects were generally less than that of TPA. Morphologically, in the presence of piperine, melanoblasts appeared to be more dendritic and the cell bodies were flatter and smaller.
• Human melanocytes used in this experiment were derived from induced differentiation of human foetal melanoblasts.
• the key character of human melanocytes that is different from its precursor melanoblasts is their ability to synthesise melanin.
• Melanin is a valid marker for melanocytes.
• the cell pellet of human melanocytes exhibits a characteristic brown to black colour, whereas human melanoblasts cannot produce melanin thus devoid of brown or black colour in the cell pellet.
• subconfluent human melanocytes maintained in a ⁇ 100 mm petri dish were subcultured into two 24-well plates (Falcon) using basic culture medium of RPMI 1640 supplemented with FBS (10%), bFGF (100 pM), CT (1 nM) and endothelin 1 (1 nM).
• the initial plating density was 20,000 cells/cm 2 (38,200 cells/well) with each well containing 1000 ⁇ l medium.
• piperine in 500 ⁇ l medium was added into wells to made up final concentrations of 0, 1, 5, 10 and 100 ⁇ M.
• subconfluent human melanocytes were subcultured in a ⁇ 60 mm petri dishes (28 cm 2 , Falcon) with RPMI 1640 basic medium supplemented with FBS (10%), CT (1 nM), bFGF (100 pM) and endothelin 1 (1 nM).
• the initial plating density was 10,000 cells/cm 2 , with 5 ml medium per dish.
• Cells were incubated for 2 - 3 hours in 10% CO 2 , humidified atmosphere, at 37°C, followed by addition of piperine solution in to the dishes, making the final concentrations of 0, 1, 5, 10 and 100 ⁇ M.
• Cells in the above supplemented medium lacking endothelin 1 were also set up as a negative control.
• Figure 7 delineates the effects of piperine on the growth of human melanocytes cultured in 24-well plate.
• Piperine at the concentrations of 5 and 10 ⁇ M markedly stimulates the growth of these pigmented cells, with 36% more cells yielded when the culture was under the influence of 10 ⁇ M piperine for 5 days.
• piperine exerted inhibitory effect on the growth of these cells.
• TPA at 20 nM was not able to stimulate cell growth in our culture system, a result that is of great difference with that observed in human melanoblasts.
• Table 5 shows the effects of piperine on the growth of human melanocytes cultured in petri dishes. It is conspicuous that in the presence of ET1 (1nM), piperine at the concentrations of 5 and 10 ⁇ M significantly stimulated the growth of human melanocytes, with cell number over twice as many as that of ET1 (1nM) control when this melanocyte cell type was exposed to 5 ⁇ M piperine for 5 days. This result was consistent with that obtained from the 24-well plate experiments, and it served to confirm that the stimulatory effects observed by SRB assay were indeed due to increased cell number rather than augmentation of protein production alone.

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• 1998
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